Experiment of oral mucosa epithelial cells cultured on small intestinal submucosa in vitro / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To explore an effective method to culture oral mucosa epithelial cells (OMECs) of canine in vitro, and to observe the biological characteristics of OMECs growing on small intestinal submucosa (SIS) in order to provide the experimental basis for epithelium tissue engineering.</p><p><b>METHODS</b>The primary OMECs were cultivated with DKSFM (defined keratinocyte serum free medium) containing 6% fetal bovine serum (FBS). The morphological characteristics and the growth curve of OMECs were observed. The expressions of OMECs marker (CK19) were examined by immunocytochemistry. The 2nd passage of OMECs were seeded on SIS, OMECs co-cultured with SIS were observed by hematoxylin-eosin staining, immunohistochemical staining, and scanning electron microscope (SEM).</p><p><b>RESULTS</b>OMECs were grown well in DKSFM. Immunohistochemical staining of the 2nd passage cultured canine OMECs with broadly reacting anti-cytokeratin anyibodies (CK19) was positive. OMECs formed a single layer on the surface of SIS, and eight days later the cells were polygong and arranged like slabstone.</p><p><b>CONCLUSION</b>Culture of canine OMECs in DKSFM containing 6% FBS is a simple and feasible method. SIS has good biocompatibility, it is a kind of good bioscafold in the tissue-engineered epithelium.</p>

Mesenchymal stem cells transplantation mildly ameliorates experimental diabetic nephropathy in rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Diabetic nephropathy is a common complication of diabetes mellitus. This study aimed to explore whether mesenchymal stem cells (MSCs) transplantation could attenuate diabetic nephropathy in experimental diabetic rats.</p><p><b>METHODS</b>Sprague-Dawley rats received a single intraperitoneal injection of streptozotocin (STZ) (60 mg/kg). Diabetic rats were randomized to four groups: diabetes control group (DC), ciclosporin A group (CsA), MSC group, and MSC + CsA group (MSCA). Bone marrow mesenchymal stem cells were cultured, identified and labeled by 5-bromo-2'-deoxyuridine (BrdU) in vitro. Then they were transplanted to diabetic rats via introcardiac infusion. Ciclosporin A was administered daily at 5 mg/kg. At 1, 2, 4, 8 weeks after transplantation, random blood glucose, urine albumin/creatinine ratio (Alb/Cr), endogenous creatinine clearance rate and renal mass index were tested. Renal morphology and labeled cells were examined.</p><p><b>RESULTS</b>Cultured MSCs expressed mesenchymal cell phenotype, and could be multidifferentiated to osteogenic and adipogenic cells. Labeled MSCs could be detected in the kidney of nephropathic rats, mainly in renal interstitium, but they did not propagate after engrafting in kidney. Over the course of the experiment, MSCA group showed a significant decrease in blood glucose compared with MSC group, CsA group and DC group (P < 0.05, respectively). The Alb/Cr in MSCA group and MSC group were significantly lower than CsA group and DC group (P < 0.05). And the Alb/Cr in MSCA group showed a significant decrease compared with MSC group (0.74 vs 0.84, P < 0.05). There was a significant difference in renal mass index between the MSCA group and DC group (5.66 vs 6.37, P < 0.05). No significant difference was found in creatinine clearance rate among 4 groups (P > 0.05). Treatment with MSC + CsA significantly ameliorated the morphology of diabetic kidney.</p><p><b>CONCLUSION</b>MSC could mildly ameliorate diabetic nephropathy by decreasing blood glucose, Alb/Cr ratio and renal mass index.</p>

The possible role of Myosin light chain in myoblast proliferation

Skeletal muscles have the potential to regenerate by activation of quiescent satellite cells, however, the molecular signature that governs satellite cells during muscle regeneration is not well defined. Myosin light chains (Myls) are sarcomere-related proteins as traditional regulator of muscle contraction. In this report, we studied the possible role of Myl in the proliferation of skeletal muscle-derived myoblasts. Compared to diaphragm-derived myoblasts, the extraocular muscle-derived myoblasts with lower levels of Myl proliferated faster, maintained a longer proliferation phase, and formed more final myotubes. It was found that blockading Myl with anti-Myl antibody or knockdown of Myll by siRNA targeted against Myll could enhance the myoblast proliferation and delay the differentiation of myoblasts. Our results suggested that Myl, likely Myll, can negatively affect myoblast proliferation by facilitating myoblast withdrawal from cell cycle and differentiation.

Effects of hypoxia on human placental decidua basalis-mesenchymal stem cells proliferation, apoptosis and VEGF expression / 生理学报

Human placental decidua basalis-mesenchymal stem cells (PDB-MSCs) are multipotent cells from the human term placenta, which are ethically conducive, easily accessible and high-yielding source. PDB-MSCs can differentiate into adipogenic, osteogenic and neurogenic cells under appropriate conditions, which may be an attractive and alternative source of seed cells for tissue engineering. To investigate the effect of hypoxia (1% O2) on human PDB-MSCs and the expression of cytokine, PDB-MSCs were isolated from human placenta by density gradient centrifugation and cultured in the Dulbecco's modified Eagle's medium-high glucose (DMEM-HG) containing 10% fetal bovine serum (FBS), and the fifth passage of PDB-MSCs were taken. PDB-MSCs were divided into 4 groups according to the concentrations of O2 and FBS: 20% O2, 10% FBS; 20% O2, 0% FBS; 1% O2, 10% FBS; 1% O2, 0% FBS. The proliferation and apoptosis of PDB-MSCs were detected by MTT and flow cytometric analysis at the time points of 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h, respectively. Vascular endothelial growth factor (VEGF) released from PDB-MSCs was detected by enzyme-linked immunosorbent assay (ELISA) at the same time points. The results showed that hypoxia enhanced the proliferation of PDB-MSCs at 12 h under the condition of 10% FBS, while at 24 h under the condition of 0% FBS (P<0.01, n=3). In normoxia, the cells cultured in 10% FBS displayed a significant proliferation compared to those cultured in 0% FBS. However, in hypoxia, the number of cells cultured in 0% FBS (serum deprivation) increased significantly compared to that cultured in 10% FBS at 24 h and 96 h respectively (P<0.05, P<0.01, n=3). With the flow cytometric analysis of cell apoptosis under the condition of hypoxia and serum deprivation, we found that hypoxia and serum deprivation did not induce PDB-MSCs apoptosis (P>0.05, n=3). This conclusion may relate to the expression of VEGF which needs further research. In conclusion, the results obtained indicate that PDB-MSCs are able to bear hypoxia and serum deprivation, suggesting that PDB-MSCs can be used as seed cells for ischemia related tissue engineering.

The experiment of porcine keratinocytes cultured on porcine small intestinal submucosa in vitro / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of acellular porcine small intestinal submucosa (SIS as bioscaffold of tissue engineering skin.</p><p><b>METHODS</b>The second passage keratinocytes were seeded on SIS, after seeded for 11, 13, 15, 17 days, the keratinocytes/SIS composites were observed by dye directly, histopathology, immunohistochemical studies with monoclonal antibodies against laminin and transmission electron microscope (TEM).</p><p><b>RESULTS</b>At eleventh day, keratinocytes were growth very well on the surface of SIS, there are 2-3 cell layers on partial of the SIS surface, the continued expression of laminin can be detected between the keratinocytes and the surface of SIS. After 13, 15, 17 days this stratified structure increased, cells contact more closely, the tonofibrils in cells, desmosome between cells and the basal membrane between the keratinocytes and the surface of SIS can be seen with TEM.</p><p><b>CONCLUSIONS</b>SIS is a kind of good bioscaffold in the culture of porcine keratinocytes in vitro.</p>

Preparation of xenogenic corneal stroma and its cytoconsistency / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the possibility that using the bovine corneal stroma to provide a suitable carrier on which the cells can grow for tissue engineering cornea.</p><p><b>METHODS</b>Nine fresh bovine corneas were selected. Each cornea was cut into 2 pieces, and exposed to 0.25% trypsinase for various lengths of time (20 minutes, 40 minutes, and 60 minutes) to get the stroma part with least cells and maintaining the collagen fibers arrangement. Samples obtained from each group were examined with scanning electron microscopy and HE staining. The left ones were freeze-dried and sterilized. The various concentrations of extraction were used to cultivate human fibroblasts, and a 3-(4,5-dimethylthiazol-2-yl)-2, (MTT)-based colorimetric assay was taken to evaluate the exhistance of 5-diphenyltetrazolium bromide cytotoxinic effects. Then the proper corneal stroma was used as a carrier to cultivated the rabbit corneal limbal cells which were planted on it in a concentration of 2 x 10(5)/cm2 in vitro. The cell-carrier samples were sent for scanning electron microscopy and HE staining.</p><p><b>RESULTS</b>The corneal stroma had the least cells in the group acted with typsin for 60 minutes, while the collagen fibers arrangement was not so orderly as before. The extractions showed no significant difference in cell culture, and no obviously harmful effect on the cell growth. The rabbit corneal limbal cells presented a stratified growth on the bovine corneal stroma.</p><p><b>CONCLUSION</b>The bovine corneal stroma without cells prepared using the typsin and lyophilization can be a suitable carrier for cell culture in vitro.</p>

Histological evaluation of direct pulp capping with a self-etching adhesive and calcium hydroxide / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the beagles' pulp response following direct pulp capping with Clearfil SE BOND (SB).</p><p><b>METHODS</b>130 sound teeth were used. 120 had their pulps mechanically exposed and were divided in two groups. In group A, teeth were capped with SB. In group B, teeth were capped with calcium hydroxide (CH). The left 10 teeth were used as control. After 7, 30 and 90 days, the teeth were extracted and processed for light microscopical examination.</p><p><b>RESULTS</b>In 7 day observation period, inflammatory reaction in SB group was slighter than that of CH group, but the difference was statistical insignificant. In the 30 day and 90 day observation period, inflammatory reaction was slight in both groups, but specimens with dentin bridge formation was significantly less in SB group than in CH group (P < 0.05).</p><p><b>CONCLUSION</b>SB showed acceptable biocompatibility with pulp, but its ability to induce hard tissue barrier on pulp exposure is weaker than CH.</p>

Ectopic osteogenesis of tissue engineered bone with slow-released bioactive factors / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the ectopic osteogenesis of tissue engineered bone with recombinant human bone morphogenetic protein/transforming growth factor-beta (rhBMP/TGF-beta) or WO-1 slow-released factors.</p><p><b>METHODS</b>Partial demineralized freeze-dried bone (PDFDB) of pig was used as scaffold material. rhBMP/TGF-beta or WO-1 were pre-coated on the surface of material by means of vacuum negative pressure absorption, and then coated with polylactic acid (PLA) to make slow-released material. There were six group: PDFDB material (group A); PDFDB combined with osteoblasts (group B); PDFDB material with rhBMP/TGF-beta slow-released system (group C); PDFDB material combined with rhBMP/TGF-beta slow-released system osteoblasts (group D); PDFDB with WO-1 slow-released system (group E); PDFDB material combined with WO-1 slow-released system and osteoblasts (group F) were implanted in bilateral lower limbs of 36 Newzealand rabbits respectively (6 rabbits in each groups). Histological, histochemical and biochemical analysis were detected 2, 4, 6, 8 weeks after operation.</p><p><b>RESULTS</b>Within the observation periods, no osteogenesis was observed in group A. The osteogenesis in group B, D, F were superior to that of group C and E (P < 0.05). The osteogenetic activity in group C and E was delayed. The quantity and quality of osteogenesis in group D and F were 2 weeks ahead of time compared with group B, and 4 weeks to that of group C and E. The newborn calcification content was superior to that of group A, C, and E (P < 0.05).</p><p><b>CONCLUSIONS</b>The osteogenesis of PDFDB materials with BMP/TGF-beta or WO-1 is slower than that of which combined with osteoblasts. Simple material PDFDB has no ectopic osteogenesis.</p>

Reconstitution of telomerase activity in human embryonic tendon cells transfected by ptsA58H plasmid / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the feasibility of life span extension of transformed human embryonic tendon cells (THETC) by reconstitution of the telomerase activity.</p><p><b>METHODS</b>THETC were transfected by pGRN145 plasmid containing the human telomerase reverse transcriptase (hTERT) cNDA in vitro by molecular cloning technique. The biological characteristics of transfected cells were detected and compared by morphological observation, plate cloning efficiency, soft agar culture, growth curve of cells cultured in different conditions, immunohistochemistry, telomerase activity assay by telomeric repeat amplification protocol (TRAP).</p><p><b>RESULTS</b>The THETC transfected by pGRN145 plasmid (telT) could express the telomerase activity with extension of life span. The telT maintained the original characteristics of temperature-dependant and serum-dependant, as well as secretion of type I collagen normally and without tendency of malignant transformation.</p><p><b>CONCLUSIONS</b>The life span of THETC can be prolonged by reconstitution of telomerase activity, which provides the novel experimental methods to establish the standard cells line.</p>